A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

A splicing isoform of GPR56 mediates microglial synaptic refinement via phosphatidylserine binding.

Developmental synaptic remodeling is important for the formation of precise neural circuitry, and its disruption has been linked to neurodevelopmental disorders such as autism and schizophrenia. Microglia prune synapses, but integration of this synapse pruning with overlapping and concurrent neurodevelopmental processes, remains elusive. Adhesion G protein-coupled receptor ADGRG1/GPR56 controls multiple aspects of brain development in a cell type-specific manner: In neural progenitor cells, GPR56 regulates cortical lamination, whereas in oligodendrocyte progenitor cells, GPR56 controls developmental myelination and myelin repair. Here, we show that microglial GPR56 maintains appropriate synaptic numbers in several brain regions in a time- and circuit-dependent fashion. Phosphatidylserine (PS) on presynaptic elements binds GPR56 in a domain-specific manner, and microglia-specific deletion of Gpr56 leads to increased synapses as a result of reduced microglial engulfment of PS+ presynaptic inputs. Remarkably, a particular alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning. Our present data provide a ligand- and isoform-specific mechanism underlying microglial GPR56-mediated synapse pruning in the context of complex neurodevelopmental processes.

GPR56 S4 variant is required for microglia-mediated synaptic pruning.

ADGRG1 (also called GPR56) plays critical roles in brain development and wiring, including cortical lamination, central nervous system (CNS) myelination, and developmental synaptic refinement. However, the underlying mechanism(s) in mediating such diverse functions is not fully understood. Here, we investigate the function of one specific alternative splicing isoform, the GPR56 splice variant 4 (S4), to test the hypothesis that alternative splicing variants of GPR56 in part support its different functions. We created a new transgenic mouse line, Gpr56∆S4 , using CRISPR/Cas9, in which GPR56 S4 was deleted. Detailed phenotype analyses show that Gpr56∆S4 mice manifest no deficits in cortical architecture and CNS myelination compared to controls. Excitingly, they present significantly increased synapse densities, decreased synapse engulfment by microglia, and impaired eye-segregation. Taken together, our findings support that the GPR56 S4 variant is dispensable for cortical development and CNS myelination but is essential for microglia-mediated synaptic pruning.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Cell type-specific evaluation of ADGRG1/GPR56 function in developmental central nervous system myelination.

Myelination of axons in the central nervous system (CNS) is a concerted effort between many cell types, resulting in significant cross-talk and communication among cells. Adhesion G protein-coupled receptor ADGRG1 (GPR56) is expressed in all major glial cells and regulates a wide variety of physiological processes by mediating cell-cell and cell-matrix communications. Previous literature has demonstrated the requirement of ADGRG1 in oligodendrocyte precursor cells (OPCs) during developmental myelination. However, it is unknown if ADGRG1 is responsible for myelin formation in a cell-type-specific manner. To that end, here we profiled myelin status in response to deletion of Adgrg1 specifically in OPCs, microglia, astrocytes, and neurons. Interestingly, we find that knocking out Adgrg1 in OPCs significantly decreases OPC proliferation and reduced number of myelinated axons. However, deleting Adgrg1 in microglia, astrocytes, and neurons does not impact developmental myelination. These data support an autonomous functional role for Adgrg1 in OPCs related to myelination.

Association of Maternal Immune Activation during Pregnancy and Neurologic Outcomes in Offspring.

OBJECTIVE: To evaluate neurologic morbidity among offspring during their first year of life in association with prenatal maternal immune activation (MIA), using an inclusive definition. STUDY DESIGN: This retrospective cohort study included singletons born in California between 2011 and 2017. MIA was defined by International Classification of Diseases diagnosis of infection, autoimmune disorder, allergy, asthma, atherosclerosis, or malignancy during pregnancy. Neurologic morbidity in infants was defined by International Classification of Diseases diagnosis of intraventricular hemorrhage, periventricular leukomalacia, seizures, abnormal neurologic examination, or abnormal neurologic imaging. Outcomes of delayed developmental milestones during the first year of life were also explored. Risk of neurologic morbidity in offspring was approximated for women with and without MIA using log link binary regression. RESULTS: Demographic characteristics among 3 004 166 mother-infant dyads with or without MIA were similar in both groups. Rate of preterm delivery in mothers with MIA (9.4%) was significantly higher than those without MIA (5.6%). Infants of mothers with MIA were more likely to experience neurologic morbidities across all gestational ages. Adjusted relative risk (95% CI) in the exposed infants was 2.0 (1.9-2.1) for abnormal neurologic examination; 1.6 (1.5-1.7) for seizures, and 1.6 (1.4-1.8) for periventricular leukomalacia. CONCLUSIONS: Our results demonstrate that MIA during pregnancy may be associated with considerably higher risk of neurologic morbidity in offspring.

Adhesion G protein-coupled receptors in nervous system development and disease.

Members of the adhesion G protein-coupled receptor (aGPCR) class have emerged as crucial regulators of nervous system development, with important implications for human health and disease. In this Review, we discuss the current understanding of aGPCR functions during key steps in neural development, including cortical patterning, dendrite and synapse formation, and myelination. We focus on aGPCR modulation of cell-cell and cell-matrix interactions and signalling to control these varied aspects of neural development, and we discuss how impaired aGPCR function leads to neurological disease. We further highlight the emerging hypothesis that aGPCRs can be mechanically activated and the implications of this property in the nervous system.

GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist.

Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.

Overlap of polymicrogyria, hydrocephalus, and Joubert syndrome in a family with novel truncating mutations in ADGRG1/GPR56 and KIAA0556.

Genetic mutations associated with brain malformations can lead to a spectrum of severity and it is often difficult to determine whether there are additional pathogenic variants contributing to the phenotype. Here, we present a family affected by a severe brain malformation including bilateral polymicrogyria, hydrocephalus, patchy white matter signal changes, and cerebellar and pontine hypoplasia with elongated cerebellar peduncles leading to the molar tooth sign. While the malformation is reminiscent of bilateral frontoparietal polymicrogyria (BFPP), the phenotype is more severe than previously reported and also includes features of Joubert syndrome (JBTS). Via exome sequencing, we identified homozygous truncating mutations in both ADGRG1/GPR56 and KIAA0556, which are known to cause BFPP and mild brain-specific JBTS, respectively. This study shows how two independent mutations can interact leading to complex brain malformations.

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve ß-cell function.

AIMS: G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on ß-cell function. METHODS: GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on ß-cell proliferation, apoptosis, intracellular calcium ([Ca2+]i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56-/- mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56-/- mice. RESULTS: Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet ß-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected ß-cells from cytokine-induced apoptosis, triggered increases in [Ca2+]i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56-/- islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation. CONCLUSION: We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

Adhesion G-protein coupled receptors and extracellular matrix proteins: Roles in myelination and glial cell development.

Adhesion G protein-coupled receptors (aGPCRs) are a large family of transmembrane proteins that play important roles in many processes during development, primarily through cell-cell and cell-extracellular matrix (ECM) interactions. In the nervous system, they have been linked to the complex process of myelination, both in the central and peripheral nervous system. GPR126 is essential in Schwann cell-mediated myelination in the peripheral nervous system (PNS), while GPR56 is involved in oligodendrocyte development central nervous system (CNS) myelination. VLGR1 is another aGPCR that is associated with the expression of myelin-associated glycoprotein (MAG) which has inhibitory effects on the process of nerve repair. The ECM is composed of a vast array of structural proteins, three of which interact specifically with aGPCRs: collagen III/GPR56, collagen IV/GPR126, and laminin-211/GPR126. As druggable targets, aGPCRs are valuable in their ability to unlock treatment for a wide variety of currently debilitating myelin disorders. Developmental Dynamics 246:275-284, 2017. © 2016 Wiley Periodicals, Inc.

Gpr126/Adgrg6 Has Schwann Cell Autonomous and Nonautonomous Functions in Peripheral Nerve Injury and Repair.

Schwann cells (SCs) are essential for proper peripheral nerve development and repair, although the mechanisms regulating these processes are incompletely understood. We previously showed that the adhesion G protein-coupled receptor Gpr126/Adgrg6 is essential for SC development and myelination. Interestingly, the expression of Gpr126 is maintained in adult SCs, suggestive of a function in the mature nerve. We therefore investigated the role of Gpr126 in nerve repair by studying an inducible SC-specific Gpr126 knock-out mouse model. Here, we show that remyelination is severely delayed after nerve-crush injury. Moreover, we also observe noncell-autonomous defects in macrophage recruitment and axon regeneration in injured nerves following loss of Gpr126 in SCs. This work demonstrates that Gpr126 has critical SC-autonomous and SC-nonautonomous functions in remyelination and peripheral nerve repair. SIGNIFICANCE STATEMENT: Lack of robust remyelination represents one of the major barriers to recovery of neurological functions in disease or following injury in many disorders of the nervous system. Here we show that the adhesion class G protein-coupled receptor (GPCR) Gpr126/Adgrg6 is required for remyelination, macrophage recruitment, and axon regeneration following nerve injury. At least 30% of all approved drugs target GPCRs; thus, Gpr126 represents an attractive potential target to stimulate repair in myelin disease or following nerve injury.

Novel mutation in CNTNAP1 results in congenital hypomyelinating neuropathy.

INTRODUCTION: Congenital hypomyelinating neuropathy (CHN) is a rare congenital neuropathy that presents in the neonatal period and has been linked previously to mutations in several genes associated with myelination. A recent study has linked 4 homozygous frameshift mutations in the contactin-associated protein 1 (CNTNAP1) gene with this condition. METHODS: We report a neonate with CHN who was found to have absent sensory nerve and compound muscle action potentials and hypomyelination on nerve biopsy. RESULTS: On whole exome sequencing, we identified a novel CNTNAP1 homozygous missense mutation (p.Arg388Pro) in the proband, and both parents were carriers. Molecular modeling suggests that this variant disrupts a ß-strand to cause an unstable structure and likely significant changes in protein function. CONCLUSIONS: This report links a missense CNTNAP1 variant to the disease phenotype previously associated only with frameshift mutations. Muscle Nerve 55: 761-765, 2017.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

Adhesion GPCRs as Novel Actors in Neural and Glial Cell Functions: From Synaptogenesis to Myelination.

Adhesion G-protein-coupled receptors (aGPCRs) are emerging as key regulators of nervous system development and health. aGPCRs can regulate many aspects of neural development, including cell signaling, cell-cell and cell-matrix interactions, and, potentially, mechanosensation. Here, we specifically focus on the roles of several aGPCRs in synapse biology, dendritogenesis, and myelinating glial cell development. The lessons learned from these examples may be extrapolated to other contexts in the nervous system and beyond.

Expression of Heme Oxygenase-1 and Leukemia Inhibitory Factor in Maternal Plasma and Placental Tissue in a Lipopolysaccharide-Induced Late Pregnancy Preterm Birth Mouse Model.

OBJECTIVE: To investigate the expression of heme oxygenase-1 (HO-1) and leukemia inhibitory factor (LIF) in maternal plasma and placental tissue in intrauterine infection-induced preterm birth. STUDY DESIGN: Using a mouse model of intrauterine infection in preterm birth, we used magnetic beads to extract normal pregnant mouse spleen Treg cells, injecting them into lipopolysaccharide (LPS)-treated pregnant mice. The subjects were divided into 4 groups: control group, LPS group, LPS+PBS group, and LPS+Treg group. RT-PCR and Western blot were used to evaluate HO-1, LIF mRNA, and protein levels in the placenta. ELISA was used to detect these parameters in the peripheral blood of pregnant mice. RESULTS: The expression of HO-1 and LIF in the placenta of the LPS group was significantly decreased when compared to that of the control group (p<0.05). Serum HO-1 and LIF levels were higher than in the control group (p<0.05). In the Treg cell-treated group placental tissue HO-1 and LIF expression were significantly higher than in the LPS group (p<0.05), and serum HO-1 and LIF expression were significantly lower than in the LPS group (p<0.05). CONCLUSION: HO-1 and LIF participate with Treg cells in the maternal-fetal interface, producing a unique immune microenvironment.

Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.

The peripheral nervous system (PNS) has remarkable regenerative abilities after injury. Successful PNS regeneration relies on both injured axons and non-neuronal cells, including Schwann cells and immune cells. Macrophages are the most notable immune cells that play key roles in PNS injury and repair. Upon peripheral nerve injury, a large number of macrophages are accumulated at the injury sites, where they not only contribute to Wallerian degeneration, but also are educated by the local microenvironment and polarized to an anti-inflammatory phenotype (M2), thus contributing to axonal regeneration. Significant progress has been made in understanding how macrophages are educated and polarized in the injured microenvironment as well as how they contribute to axonal regeneration. Following the discussion on the main properties of macrophages and their phenotypes, in this review, we will summarize the current knowledge regarding the mechanisms of macrophage infiltration after PNS injury. Moreover, we will discuss the recent findings elucidating how macrophages are polarized to M2 phenotype in the injured PNS microenvironment, as well as the role and underlying mechanisms of macrophages in peripheral nerve injury, Wallerian degeneration and regeneration. Furthermore, we will highlight the potential application by targeting macrophages in treating peripheral nerve injury and peripheral neuropathies.